This proposal is designed to determine the mechanism of elongation by human RNA polymerase II (RNAP II) and to elucidate combinatorial control by elongation factors. The Burton laboratory has pioneered and verified rapid chemical quench-flow kinetic methods to analyze RNAP II functional dynamics during elongation in real time (millisecond phase), using combinations of potent regulators. The Specific Aims are to: 1) characterize the RNAP II elongation control system, develop and refine a kinetic mechanism for elongation by human RNAP II, and challenge the NTP driven translocation model for RNAP II elongation; 2) determine the roles of viral hepatitis delta antigen (HDAg), human Transcription Factor IIF (TFIIF), and human TFIIS in regulation of RNAP II elongation, and use these factors as probes of the RNAP II mechanism; 3) identify mechanisms for fidelity and efficiency of RNAP II elongation; and 4) initiate rapid quench-flow studies of yeast RNAP II elongation to exploit the yeast RNAP II elongation control proteome. Kinetic analysis of human RNAP II gives novel insight into the mechanism and regulation of translocation, NTP loading, pyrophosphate release and the efficiency and fidelity of NMP incorporation.